In the development of specific diagnostic or therapeutic methods, the use of transfer systems (delivery systems) are of great importance which permit a transfer as cell-specific as possible of substances such as nucleic acid, markers or active ingredients. For this cell-specific transfer, inter alia, a system based on virus-like particles (VLP) has been developed (WO 97/19174; EP 1 270 586 B1). The basis of this system is the property of the VLP to be able, for example, to package active ingredients or nucleic acids, and then to implant them specifically into defined cells.
VLPs can be produced, for example, by recombinant expression of the main structural protein VP1 of the human polyomavirus JCV (VP1-VLPs). In contrast to VP1 expression of other polyomaviruses, the VP1-VLPs are secreted into the supernatant of the host cell cultures. For purification of the VP1-VLPs from the cell culture supernatant, a plurality of methods have already been developed. However, these are not all suitable for producing VLPs on a commercial scale (large scale). This is true in particular when the production process must be GMP-certifiable, since particularly high demands are then made on the purity of the VLPs.
For instance, Goldmann et al. (J. Virol., 1999, 73: 4465-4469) describe the purification of VP1-VLPs expressed in insect cells by density centrifugation using a 40% strength sucrose solution followed by a density centrifugation using 40% sucrose and 50% metrizamide(2-({3-(acetylamino)-5-[acetyl(methyl)amino]-2,4,6-triiodobenzoyl}amino)-2-deoxy-D-glucopyranose. This method is unsuitable not only for large scale production. Also, the VP1 proteins provided in this way are contaminated with VP1 fragments of 38 and 40 kDa.
For purification of recombinant VLPs from lysed E. coli cells, Pushko et al. (Protein Engineering, 1993, 6(8): 883-891) use an ammonium sulfate precipitation with subsequent gel permeation chromatography with the use of a Sephadex G25 column and a G100 column.
WO 92/13081 A1 discloses a purification method for isolating VLPs derived from MS-2 by fractional ammonium sulfate precipitation and subsequent isoelectric point precipitation, sucrose density centrifugation and gel permeation chromatography.
WO 2006/136566 A9 describes the purification of recombinant bacterially expressed VLPs via an anion-exchange chromatography followed by a hydroxyl apatite column and an optional gel permeation chromatography.
The methods known from this prior art are insufficiently suitable for preparing the VLPs outside a laboratory scale since they are either very complex, do not permit upscaling to an industrial method and/or do not meet the high requirements of a GMP-conforming process. The latter especially applies due to contaminants, which, inter alia, can also be enclosed in the VLPs.